Staining with nitrido-bridged osmium or ruthenium compounds

ABSTRACT

This invention relates to the staining of cells, and in particular provides a novel reagent for the detection, for example, of DNA (deoxyribonucleic acid) and polysaccharides by electron microscopy. A reagent for staining cells in accordance with the present invention comprises an aqueous solution of a nitrido-bridged complex of osmium or ruthenium and having the general formula: [M2IVN(NH3)8-2xX5-y(H2O)x]Yy. where M represents either Os or Ru, X and Y are the same or different anions selected from nitrate, halide and pseudohalide, x is 0 or 1 and y is 2 or 3.

This invention relates to the staining of cells, and in particular provides a novel reagent for the detection, for example, of DNA (deoxyribonucleic acid) and polysaccharides by electron microscopy.

In the technique of electron microscopy, previously-prepared biological specimens are commonly reacted with a staining reagent in order to increase the differential electron scattering power of the specimen constituents. In other words, the contrast of the electron micrograph of the specimen is increased. Particular reagents are known to stain particular compounds or classes of compound; for example, phosphotungstic acid is a cationic stain and tends to bind to proteins, whereas uranyl acetate is an anionic stain and is reasonably selective for nucleic acids and, to a lesser extent, proteins.

One reagent which is known to be specific for deoxyribonucleic acid (DNA) is a so-called "Feulgen" stain. The reagent is generally a Schiff's reagent which reacts with aldehyde groups liberated by hydrolysis of tissue hydroxyl groups from DNA. Typically, however, reagents of the Schiff's type give only a low contrast or give irregular results and are, therefore, unsuitable for routine work.

One reagent that has been proposed to improve on the traditional Feulgen stains for detection of DNA, and which is also active for the detection of polysaccharides by a PAS-type reaction is so-called "osmium ammine". This is a black crystalline powder which appears to show reproducible staining properties but whose structure is uncharacterised, it being apparently a mixture of compounds. In use, specimens are prepared in the usual way as far as fixation and embedding are concerned and thin sections are then submitted to mild acid hydrolysis before being floated on a solution of the reagent in water through which SO₂ has been passed. The reagent concentration, temperature and duration of the staining reaction are variable and selected according to the desired intensity and grain fineness for a given specimen.

It is an object of the present invention to provide a novel staining reagent which is at least equal in performance to "osmium ammine" and which is easy to prepare and characterise.

According to a first aspect of the invention, a reagent for staining cells comprises an aqueous solution of a nitrido-bridged complex of osmium or ruthenium.

According to a second aspect of the invention, a method for staining cells of a prepared biological specimen (as hereinbefore defined) comprises submitting said specimen firstly to mild acid hydrolysis and secondly to reaction with a staining reagent comprising an aqueous solution of a nitrido-bridged complex of osmium or ruthenium.

The general formula of the nitrido-bridged complex is

    [M.sub.2.sup.IV N(NH.sub.3).sub.8-2x X.sub.5-y (H.sub.2 O).sub.x ]Y.sub.y

where M represents either Os or Ru, X and Y are the same or different anions selected from nitrate, halide and pseudohalide, x is 0 or 1 and y is 2 or 3.

A pseudohalide is an anion of a pseudohalogen and is defined in "Advanced Inorganic Chemistry" by F. A. Cotton and G. Wilkinson, 2nd Edition, p.560, as being a molecule consisting of more than two electronegative atoms which, in the free state, resembles the halogens, the most important being (CN)₂ cyanogen, (SCN)₂ thiocyanogen, (SeCN)₂ selenocyanogen and (SCSN₃)₂ azidocarbondisulphide. In addition there are the azide N₃ ⁻ and cyanate OCN⁻ ions which have no pseudohalogen parent. The pseudohalides which we prefer to use in reagents according to the present invention are azide and, not mentioned in the above list, isothiocyanate NCS⁻.

Three classes of complex which we prefer to use in reagents according to the invention are [Os₂ N(NH₃)₈ X₂ ]Y₃, [Ru₂ N(NH₃)₈ X₂ ]Y₃ and [Ru₂ N(NH₃)₆ X₃ (H₂ O)]Y₂, where X and Y are anions as defined above. Complexes of the first type where X and Y are chloride may conveniently be prepared by heating concentrated ammonia solution with sodium chloroosmate or K₃ [Os₂ NCl₈ (H₂ O)₂ ] in a sealed reaction vessel and reacting the product with concentrated hydrochloric acid, and complexes of the first type where X and Y are anions (either the same or different) other than chloride may readily be prepared from the chloride analogues. Complexes of the second type where X and Y are chloride may be prepared by adding a solution of K₃ [Ru₂ NCl₈ (H₂ O)₂ ] to boiling concentrated aqueous ammonia and reacting the product with concentrated hydrochloric acid and complexes of the third type where X and Y are chloride may be prepared from the mother-liquor from the preparation of complexes of the second type. Complexes of both the second and third type where X and Y are anions (either the same or different) other than chloride may be prepared either by a method similar to that for the preparation of the chloride analogues or directly from the said chloride analogues.

Reagents according to the first aspect of the invention comprise a solution of a nitrido-bridged complex of ruthenium or osmium in distilled water. Typically, the concentration of such a staining solution is 1 mg of complex per milliliter of distilled water. The staining solution may be treated by bubbling SO₂ gas through it, typically for 5 minutes before use. This treatment is particularly applicable for staining polysaccharides.

Specimens are typically prepared for staining by fixing in Karnovsky fixative, a mixture of glutaraldehyde and formalin, dehydrating and embedding without osmium post fixing. Thin sections (10 mm) are cut and mounted on gold grids. Alternatively, blocks of tissue may be fixed, hydrolysed and stained before embedding and sectioning.

A typical procedure for staining polysaccharides comprises a first hydrolysis step in which a section of the specimen, prepared by fixing and sectioning as above, is floated on a solution of 1% periodic acid for one hour, a second washing step in which the hydrolysed specimen is washed twice in distilled water, each wash being of five minutes' duration, and a third staining step in which the washed specimen is submerged in the staining solution, preferably pre-treated with SO₂, for one hour. The specimen, after a final wash in distilled water, is then ready for examination in the electron microscope.

The procedure for staining DNA is typically to fix small blocks (1 mm³) of tissue in Karnovsky fixative for 11/2 hours and wash them in distilled water. The blocks are then hydrolysed with 6 N HCl for 45 minutes, washed twice in distilled water and submerged in the staining solution for 1 hour. The blocks are then washed in distilled water, dehydrated and embedded, and their sections are cut for examination without further staining. The staining solution need not be treated with SO₂.

The preparation of complexes forming reagents according to the invention will now be described by way of example, followed by testing results in the staining of glycogen and DNA compared to the performance of osmium ammine.

PREPARATION OF THE COMPLEXES Di-chloro-octa-ammine μ-nitrido di-osmium (IV) trichloride [Os₂ N(NH₃)₈ Cl₂ ]Cl₃

Method 1

Concentrated ammonia solution (0.880; 20 ml) was added to Na₂ [OsCl₆ ] (2 g) in a Carius tube and the solution was heated to 100° C. for twelve hours. The resulting yellow solution was filtered and the black precipitate washed with cold dilute aqueous ammonia until the washings were colourless. The washings and filtrate were combined, evaporated to half bulk on the steam bath, cooled, and concentrated hydrochloric acid (10 ml) added to precipitate the product. This was washed with 2 N hydrochloric acid and reprecipitated from aqueous solution with more acid. Yield of yellow crystals 55%.

Method 2

Concentrated ammonia (0.880; 5 ml) was added to a solution of K₃ [Os₂ NCl₈ (H₂ O)₂ ] (0.3 g) and the mixture heated to 100° C. in a sealed Carius tube for twelve hours. The yellow solution was treated as before. The yield was virtually quantitative. The product from both these methods appeared to be a dihydrate; the water could be removed by heating in vacuo to 80° C.

Found for dihydrate: N: 17.4; Cl: 23.5; Os: 50.5%. H₂₈ N₉ O₂ Cl₅ Os₂ requires N: 16.9; Cl: 23.8; Os: 51.5%.

Found for the anhydrous complex: N: 18.4; Cl: 24.6%. H₂₄ N₉ Cl₅ Os₂ requires N: 17.8; Cl: 25.1%.

Electronic spectrum: 265 mμ(shoulder: ε=1.3×10⁴); 242 mμ(ε=4.9×10⁴); ˜190 mμ (ε=0.7×10⁴).

Magnetic susceptibility: Xg=-2.8×10⁻⁷ cgsu (20° C.)

Molar Conductivity:

Initial values:

1.34×10⁻³ M; 350 ohm⁻¹ cm²

4.92×10⁻⁴ M; 390 ohm⁻¹ cm²

1.03×10⁻⁴ M 415 ohm⁻¹ cm²

Final value on standing: 530 ohm⁻¹ cm².

Complexes of the form [Os₂ N(NH₃)₈ X₂ ]X₃ (X=Cl, Br, I, NCS, N₃, NO₃)

These were prepared in high yield by refluxing an aqueous solution of [Os₂ N(NH₃)₈ Cl₂ ]Cl₃ with an excess of the potassium or sodium salt of X⁻ for about one hour. The products are all yellow or yellow-brown.

[Os₂ N(NH₃)₈ Br₂ ]Br₃ --Found H: 2.9; Br: 43.6; N: 14.3%. H₂₄ N₉ Br₅ Os₂ requires H: 2.6; Br: 43.0; N: 13.6%.

Magnetic susceptibility: Xg=-2.7×10⁻⁷ c.g.s.u. (20° C.)

Electronic spectrum: 272 mμ(sh) (ε=18,100); 245 mμ(ε=49,800); 197 mμ(ε=58,550)

[Os₂ N(NH₃)₈ I₂ ]I₃ Found N: 10.5; I: 54.4%. H₂₄ N₉ I₅ Os₂ requires N: 10.8; I: 54.5.

[Os₂ N(NH₃)₈ (NCS)₂ ] (NCS)₃ --Found C: 6.4; H: 3.7; N: 23.9%. C₅ H₂₄ N₁₄ S₅ Os₂ requires C: 7.3; H: 3.0; N: 24.2%.

Magnetic susceptibility: Xg=-2.4×10⁻⁷ c.g.s.u.

Electronic spectrum: 285 mμ(ε=78,860)--due to NCS⁻ ; 240 mμ(sh) (ε=21,140); 194 mμ(ε=36,120).

Molar conductivity: 1.21×10⁻⁴ M 330 ohm⁻¹ cm².

[Os₂ N(NH₃)₈ (N₃)₂ ] (N₃)₃ --Found H: 3.8; N: 44.5%. H₂₄ N₂₄ Os₂ requires H: 3.3; N: 45.4%.

[Os₂ N(NH₃)₈ (NO₃)₂ ] (NO₃)₃ was made by dissolving [Os₂ N(NH₃)₈ Cl₂ ]Cl₃ in the minimum amount of hot water and adding excess silver nitrate to the solution. Dilute nitric acid was then added and the solution boiled to coagulate the silver chloride. The solution was then filtered into excess ethanol; the precipitate thus formed was dissolved in warm water and re-precipitated with concentrated nitric acid. The product formed deep yellow crystals. (Yield 40%).

Found: N: 24.1; O: 27.6%. H₂₄ N₁₄ O₁₅ Os₂ requires N: 23.3; O: 28.5%.

Complexes of the form [Os₂ N(NH₃)₈ X₂ ]Y₃ (XorY=Br⁻, I⁻, NCS⁻, N₃ ⁻, NO₃ ⁻)

These complexes may be prepared by the addition of a strong aqueous solution of the sodium or potassium salt of Y⁻ to a cold aqueous solution of [Os₂ N(NH₃)₈ X₂ ]X₃ in the presence of a small amount of X⁻. The solubility of [Os₂ N(NH₃)₈ X₂ ]X₃ is often low in the presence of X⁻ but if the methathesis is carried out without the latter, there is a danger of some Y⁻ becoming co-ordinated. Examples of the many complexes covered by this general method are:

[Os₂ N(NH₃)₈ Cl₂ ]I₃ --Found N: 12.7; Os: 36.4%. H₂₄ N₉ Cl₂ I₃ Os₂ requires N: 12.7; Os: 38.6%.

[Os₂ N(NH₃)₈ Cl₂ ](NCS)₃ --Found C: 4.6; N: 22.0; Cl: 10.0%. H₂₄ C₃ N₁₂ Cl₂ S₃ Os₂ requires C: 4.6; N: 21.7; Cl: 9.1%.

[Os₂ N(NH₃)₈ Cl₂ ]Br₃ --Found N: 15.2; Br: 32.9; Cl: 5.0%. H₂₄ N₉ Cl₂ Br₃ Os₂ requires N: 15.0; Br: 28.5; Cl: 8.4%.

[Os₂ N(NH₃)₈ (N₃)₂ ]I₃ --Found N: 20.8; I: 37.9%. H₂₄ N₁₅ I₃ Os₂ requires N: 21.1; I: 38.2%.

[Os₂ N(NH₃)₈ (NO₃)₂ ]Cl₃ --Found N: 20.3; Cl: 13.6%. H₂₄ N₁₁ Cl₃ O₆ Os₂ requires N: 20.3; Cl: 14.0%.

[Os₂ N(NH₃)₈ (NCS)₂ ]Cl₃ --Found N: 20.8; C: 3.5; Cl: 13.8%. H₂₄ C₂ N₁₁ S₂ Cl₃ Os₂ requires N: 20.5; C: 3.2; Cl: 14.1%.

Di-chloro-octa-amineμ-nitrido diruthenium (IV) trichloride [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃

A concentrated aqueous solution of K₃ [Ru₂ NCL₈ (H₂ O)₂ ](2 g. in 20 mls. water) was slowly added to boiling aqueous ammonia (0.880-500 mls.). The turbid solution was filtered to remove a brown precipitate and the filtrate evaporated to low bulk (˜50 ml). Concentrated hydrochloric acid (5 mls.) was added to the cold solution and the product separated out as pale orange microcrystals from an orange-red mother liquor. The latter was retained for the isolation of [Ru₂ N(NH₃)₆ Cl₃ (H₂ O)]Cl₂ The orange crystals of [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃ were purified by dissolving in hot water and reprecipitating from the cold solution with concentrated NCl. Yield 30%.

Found: H: 4.9; N: 23.6; Cl: 33.2; Ru: 38.0%. H₂₄ N₉ Cl₅ Ru₂ requires H: 4.6; N: 23.8; Cl: 33.5; Ru: 38.2%.

Magnetic susceptibility: Xg=-3.8×10⁻⁷ c.g.s.u. (20° C.)

Electronic spectrum: 320 mμ(ε=1,440), 265 mμ(ε=32,020).

235 mμ(sh) (ε=9,550), 200 mμ(ε=21,710).

Molar conductivity: Initial values varied with concentration as follows:

0.98×10⁻⁴ M; 395 ohm⁻¹ cm².

3.51×10⁻⁴ M; 356 ohm⁻¹ cm².

3.85×10⁻⁴ M; 320 ohm⁻¹ cm².

9.86×10⁻⁴ M; 305 ohm⁻¹ cm².

Long standing molar conductivity rises to ˜570 ohm⁻¹ cm².

Trichloroaquo-hexammino μ-nitrido diruthenium (IV) dichloride [Ru₂ N(NH₃)₆ Cl₃ (H₂ O)]Cl₂

The orange-red mother liquor from the preparation of [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃ was evaporated to low bulk and cooled to yield deep orange crystals of the product. This was purified either by reprecipitation from a strong aqueous solution by the addition of concentrated HCl or by recrystallisation from a weaker solution containing HCl.

Found: H: 3.6; O: 3.0; N: 19.4; Cl: 34.2; Ru: 39.1%. Calculated: H: 3.9; C: 3.1; N: 19.1; Cl: 34.5; Ru: 39.4%.

Magnetic susceptibility: Xg=-2.0×10⁻⁷ c.g.s.u. (21° C.)

Electronic spectrum: 320 mμ(ε=1,500); 264 mμ(ε=30,540)

225 mμ(ε=11,240); 195 mμ(ε=24,200).

Conductivity:

Initial values:

1.15×10⁻⁴ M; 274 ohm⁻¹ cm².

2.11×10⁻⁴ M; 308 ohm⁰¹ cm².

Final value after overnight standing: ˜500 ohm⁻¹ cm².

Dibromo-octa-ammino μ-nitrido di-ruthenium (IV) tribromide [Ru₂ N(NH₃)₈ Br₂ ]Br₃

This was made in the same way as the chloro derivative but using K₃ [Ru₂ NBr₈ (H₂ O)₂ ] and hydrobromic acid. Yield 30% of orange crystals.

Found: N: 16.4; Br: 53.7; Ru: 26.6% H₂₄ N₈ Br₅ Ru₂ requires N: 16.8; Br: 53.1; Ru: 26.9%.

Magnetic susceptibility: Xg=-3.1×10⁻⁷ c.g.s.u. (21° C.)

Molar conductivity:

0.96×10⁻⁴ M Initial value 420 ohm⁻¹ cm².

1.13×10⁻⁴ M Initial value 400 ohm⁻¹ cm².

After long standing: ˜640 ohm⁻¹ cm².

A small amount of [Ru₂ N(NH₃)₆ (H₂ O)Br₃ ]Br₂ was obtained by working up the mother liquor, but the major yield was brown crystals of a more substituted complex which appears to be Ru₂ N(NH₃)₄ Br₅ (H₂ O). Analytical and conductivity data do not provide a definite distinction between the species [Ru₂ N(NH₃)₄ Br₅ (H₂ O)] and [Ru₂ N(NH₃)₄ Br₄ (H₂ O)₂ ] Br. However, the conductivity was high, even when the measurement was made immediately after the solution had been prepared. This suggested that the nitrate groupsters very weakly bonded to the metal and very labile in solution. The latter was confirmed by the addition of excess X⁻ (X=I,NCS) to a warm aqueous solution of [Ru₂ N(NH₃)₈ (NO₃)₂ ] (NO₃)₃ (no further heating was involved). The crystalline precipitates obtained analysed as octammine rather than hexammine complexes. This is a useful means of preparing substituted octammine species, especially those such as the thiocyanato complex which readily forms a hexammine.

[Ru₂ N(NH₃)₈ I₂ ]I₃ Found H: 2.5; N: 13.2; I: 62.9%. H₂₄ N₉ I₅ Ru₂ requires H: 2.8; N: 12.6: I: 64.5%.

[Ru₂ N(NH₃)₈ (NCS)₂ ] (NCS)₃ --Found C: 10.5; H: 3.9; N: 31.5%. C₅ H₂₄ N₁₄ S₅ Ru₂ requires C: 9.4; H: 3.7; N: 30.5%.

Complexes of the form [Ru₂ N(NH₃)₈ X₂ ]Y₃ (X=Cl, Br; Y=Br, I, NCS, N₃, NO₃, NO₂) were made by the addition of a cold aqueous solution of [Ru₂ N(NH₃)₈ X₂ ]X₃ containing a small amount of X⁻ to a strong solution of the potassium salts of Y⁻.

EXAMPLES

[Ru₂ N(NH₃)₈ Cl₂ ] (NCS)₃ --Found: H: 4.5; C: 5.8; Cl: 11.9; N: 27.8%. H₂₄ C₃ N₁₂ S₃ Cl₂ Ru₂ requires H: 4.1; C: 6.0; Cl: 11.9; N: 28.1%.

The former is preferred on the grounds of the low solubility (water) and relatively low conductivity (this may be due to hydrolysis or aquation).

Found: N: 9.9; Br: 55.7%. H₁₄ N₅ OBr₅ Ru₂ requires N: 10.0; Br: 56.9%. H₁₆ N₅ O₂ Br₅ Ru₂ requires N: 9.7; Br: 55.5%.

Molar conductivity: 0.50×10⁻⁴ M(H₂ O) 55 ohm⁻¹ cm²

Dinitrato octa-ammino μ-nitrido di-ruthenium (IV) trinitrate [Ru₂ (NH₃)₈ (NO₃)₂ ] (NO₃)₃

An aqueous solution of [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃ was boiled with dilute nitric acid and excess silver nitrate until the silver chloride which forms had coagulated. The solution was filtered and the filtrate boiled once more to remove any excess silver chloride, cooled and filtered into excess ethanol.

The resulting precipitate was filtered, dissolved in water and reprecipitated as orange crystals with concentrated nitric acid. (Yield 60%).

Found: N: 29.4; Ru: 30.1% H₂₄ N₁₄ O₁₅ Ru₂ requires N: 29.6; Ru: 30.5%

Electronic spectrum: 254 mμ(ε=2.3×10⁴) 209 mμ(ε=7.26×10⁴)

Molar conductivity:

1.56×10⁻⁴ M soln. 625 ohm⁻¹ cm.²

1.04×10⁻⁴ M soln. 630 ohm⁻¹ cm.²

[Ru₂ N(NH₃)₈ Cl₂ ]I₃ --Found I: 47.4%. H₂₄ N₉ Cl₂ I₃ Ru₂ requires I: 47.4%.

[Ru₂ N(NH₃)₈ Cl₂ ] (NO₃)₃ --Found H: 3.8; N: 28.6; Cl: 8.1%. H₂₄ N₁₂ O₉ Cl₂ Ru₂ requires H: 4.2; N: 29.2; Cl: 12.3%. (This was made without the presence of Cl⁻).

[Ru₂ N(NH₃)₈ Br₂ ] (NCS)₃ Found: C: 4.9; H: 3.6; N: 24.2%. H₂₄ C₃ N₁₂ S₃ Br₂ Ru₂ requires C: 5.2; H: 3.5; N: 24.5%.

Hexammine complexes

These appear to take the form [Ru₂ N(NH₃)₆ (H₂ O)X₃ ]Y₂ (X=NCS N₃, NO₂ ; Y=Cl, I). They were made by heating an aqueous solution of [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃ with excess of the potassium or sodium salt of X⁻. The solutions on cooling deposit orange-red crystals of type [Ru₂ N(NH₃)₆ X₃ (H₂ O)]X₂ (X=NCS, N₃ NO₂) in good yield.

[Ru₂ N(NH₃)₆ (NCS)₃ (H₂ O)](NCS)₂. Found: C: 8.9; N: 27.6; Ru: 32.2%. H₂ OC₅ N₁₂ OS₅ Ru₂ requires C: 9.6; N: 27.6; Ru: 32.2%.

Magnetic susceptibility: Xg=-3.7×10⁻⁷ c.g.s.u. (21° C.)

Electronic spectrum:

326 mμ(NCS⁻ band-ε=2.9×10⁴);

248 mμ(ε=2.44×10⁴); 198 mμ(ε=2.28×10⁴).

Molar conductivity:

1.04×10⁻⁴ M Initial value 210 ohm⁻¹ cm²

2.01×10⁻⁴ M Initial value 180 ohm⁻¹ cm²

After long standing 230 ohm⁻¹ cm².

[Ru₂ N(NH₃)₆ (N₃)₃ (H₂ O)] (N₃)₂ --Found H: 4.0; N: 55.9%. H₂₀ N₂₁ ORu₂ requires H: 3.7; N: 56.4%.

[Ru₂ N(NH₃)₆ (NO₂)₃ (H₂ O)] (NO₂)₂ --found H: 4.3; N: 29.8%. H₂₀ N₁₂ O₁₁ Ru₂ requires H: 3.5; N: 29.7%.

Electronic spectrum:

297 mμ(sh) (ε=5.06×10³);

259 mμ(ε=2.74×10⁴)

232 mμ(sh) (ε=1.73×10⁴);

208 mμ(ε=1.76×10⁴).

Addition of a potassium salt of Y⁻ (Y=Cl, I, NCS) to an aqueous solution of [Ru₂ N(NH₃)₆ (H₂ O)X₃ ]X₂ containing some X⁻ ion, yield the complexes [Ru₂ N(NH₃)₆ H₂ OX₃ ]Y₂.

[Ru₂ N(NH₃)₆ (H₂ O)(NCS₃ ]Cl₂ --Found C: 6.2; N: 23.4; Cl: 11.6%. H₂₀ C₃ N₁₀ CS₃ Cl₂ Ru₂ requires C: 6.2; N: 24.2; Cl: 12.2%.

[Ru₂ N(NH₃)₆ (H₂ O)(N₃)₃ ]Cl₂ --Found N: 41.5; Cl: 13.3%. H₂₀ N₁₆ OCl₂ Ru₂ requires N: 41.9; Cl: 13.3%.

[Ru₂ N(NH₃)₆ H₂ CCl₃ ](NCS)₂ --Found C: 4.5; H: 3.4; N: 22.7%. C₂ H₂ OH₉ OCl₃ S₂ Ru₂ requires C: 4.3; H: 3.6; N: 22.6%.

[Ru₂ N(NH₃)₆ H₂ O(NO₂)₃ ]Cl₂ --Found N: 26.5; Cl: 13.5%. H₂₀ N₁₀ O₇ Cl₂ Ru₂ requires N: 25.7; Cl: 13.0%.

    ______________________________________                                         TEST RESULTS                                                                               Treatment        Stain-                                            Complex       Compound  Specimen     ing                                       ______________________________________                                         Glycogen                                                                       Staining                                                                       Osmium ammine 5 min. SO.sub.2                                                                          1% PA 1hr    5+                                                      --        1% PA 1hr then                                                                              --                                                                SO.sub.2 /H.sub.2 O 30 min                                           5 min. SO.sub.2                                                                          --           --                                        [Os.sub.2 N(NH.sub.3).sub.8 Cl.sub.2 ]Br.sub.3                                               5 min. SO.sub.2                                                                          1% PA 1hr    5+                                                      --        "            --                                                      5 min. SO.sub.2                                                                          --           --                                        [Os.sub.2 N(NH.sub.3).sub.8 Cl.sub.2 ](NO.sub.3).sub.3                                       "         1% PA 1hr    5+                                                      --        1% PA 1hr then                                                                              5+                                                                SO.sub.2 /H.sub.2 O 30 min                             [Os.sub.2 N(NH.sub.3).sub.8 Cl.sub.2 ]I.sub.3                                                --        "            2+                                        DNA STAINING                                                                   [Os.sub.2 N(NH.sub.3).sub.8 Cl.sub.2 ](NO.sub.3).sub.3                                       5 min. SO.sub.2                                                                          6N HCl 1hr   3+                                        ______________________________________                                     

We claim:
 1. A method for staining cells of a prepared biological specimen which comprises submitting said specimen firstly to mild acid hydrolysis and secondly to reaction with a staining reagent comprising an aqueous solution of a nitrido-bridged complex of osmium or ruthenium having the general formula

    [M.sub.2.sup.IV N(NH.sub.3).sub.8-2x X.sub.5-y (H.sub.2 O).sub.x ]Y.sub.y

where M represents either Os or Ru, X and Y are the same or different anions selected from the group consisting of nitrate, halide and psuedohalide, x is 0 or 1 and y is 2 or
 3. 2. A method according to claim 1 wherein the said complex has the formula(i) [Os₂ N(NH₃)₈ X₂ ]Y₃, (ii) [Ru₂ N(NH₃)₈ X₂ ]Y₃, or (iii) [Ru₂ N(NH₃)₆ X₃ (H₂ O)]Y₂.
 3. A method according to claim 2 wherein X and Y in formula (i) and formula (ii) are chloride.
 4. A method according to claim 2 wherein the complex has the formula:(iv) [Os₂ N(NH₃)₈ Cl₂ ]Cl₃, (v) [Ru₂ N(NH₃)₈ Cl₂ ]Cl₃, (vi) [Ru₂ N(NH₃)₆ Cl₃ (H₂ O)]Cl₃, or (vii) [Ru₂ N(NH₃)₈ Br₂ ]Br₃. 